蛇床子催眠活性组分对对氯苯丙氨酸致失眠大鼠海马钟基因与氨基酸类神经递质表达的影响

Effects of hypnotic active components of Cnidii Fructus on expression of hippocampal Clock genes and amino acid neurotransmitters in PCPA insomnia rats

目的观察蛇床子催眠活性组分(SCZ)对失眠大鼠海马神经递质及钟基因表达的影响,探讨其催眠作用机制。方法采用ip对氯苯丙氨酸(PCPA)建立大鼠失眠模型,ig给予低(25 g/kg)、中(50 g/kg)、高(100 g/kg)剂量SCZ,并设阳性对照(地西泮)组,给药3 d,免疫组织化学法检测大鼠海马齿状回γ-氨基丁酸(GABA)和谷氨酸(Glu)受体蛋白的表达;高效液相色谱法和免疫组化法检测大鼠海马GABA和Glu含量的变化;实时荧光定量PCR检测大鼠海马Clock、Bmal1、Cry1、Cry2、Per1、Per2、Per3等生物钟基因的表达水平。结果免疫组化结果显示,与对照组比较,模型组大鼠海马齿状回GABA表达水平显著降低,Glu表达水平显著升高(P<0.05、0.01);与模型组比较,SCZ中、高剂量组大鼠海马齿状回GABA表达水平显著升高,Glu表达水平显著降低(P<0.01)。HPLC结果显示,与对照组比较,模型组大鼠海马GABA表达水平显著降低,Glu表达水平显著升高(P<0.05、0.01);与模型组比较,SCZ中、高剂量组大鼠海马组织Glu表达水平显著降低(P<0.05),SCZ高、低剂量组大鼠GABA表达水平显著升高(P<0.01)。生物钟基因表达水平检测结果显示,与对照组比较,模型组大鼠Clock、Bmal1基因表达水平显著升高(P<0.05),Cry1、Per1、Per2基因表达水平显著降低(P<0.05、0.01)。与模型组比较,SCZ中剂量组大鼠Clock、Bmal1基因表达水平显著降低(P<0.01);SCZ高剂量组大鼠Cry1、Per1、Per2基因表达水平显著增高(P<0.05、0.01),SCZ低剂量组大鼠Per1基因表达水平显著增高(P<0.01)。结论蛇床子催眠活性组分通过降低海马Clock、Bmal1表达,提高Cry1、Per1、Per2基因的表达水平,增加抑制性、减少兴奋性氨基酸类神经递质的表达,从而调节睡眠-觉醒周期,达到治疗失眠的效果。

Objective To study the hypnotic mechanism of Cnidii Fructus hypnotic active constituent（SCZ） on the expression of clock genes and hippocampal neurotransmitters in insomnia rats. Methods The insomnia model was established by intraperitoneal injection of para-chlorophenylalanine（PCPA）. SCZ at low（25 g/kg）, medium（50 g/kg）, and high（100 g/kg） conentrations were ig administrated respectively at the same time with diazepam as positive group, intragastric administration of PCPA was performed in insomnia rats for three days, The expression levels of Clock, Bmal1, Cry1, Cry2, Per1, Per2, and Per3 were detected by real-time PCR. The expression levels of γ-aminobutyric acid（GABA） and glutamic acid（Glu） in hippocampus dentate gyrus were detected by immunohistochemistry. The changes of contents of GABA and Glu were determinated by immunohistochemistry and high performance liquid chromatography. Results The results of immunohistochemistry showed that the expression of GABA in the dentate gyrus of the hippocampus of rats in the model group was significantly reduced compared with the control group, and the expression level of Glu was significantly increased（P〈0.05, 0.01）. Compared with the model group, the expression level of GABA in SCZ medium and high dose group was significantly increased, and Glu expression was reduced significantly（P〈0.01）. HPLC results showed that the GABA expression level in the hippocampus of the model group was significantly decreased compared with the control group, and the expression level of Glu was significantly increased（P〈0.05, 0.01）. Compared with the model group, the expression level of Glu in hippocampus of rats in SCZ medium and high dose group was significantly decreased（P〈0.05）, and the expression of GABA in rats with high and low doses of SCZ was significantly increased（P〈0.01）. The results of Clock gene expression test showed that the expression of Clock and Bmal1 gene in the model group was significantly increased（P〈0.05） compared with the control group, and the expression levels of Cry1, Per1, and Per2 genes were significantly decreased（P〈0.05, 0.01）. Compared with the model group, the expression of Clock and Bmal1 gene in the SCZ medium dose group was significantly decreased（P〈0.01）; The expression levels of Cry1, Per1, and Per2 in the SCZ high dose group were significantly increased（P〈0.05, 0.01）. The Per1 gene expression in the SCZ low-dose group was significantly increased（P〈0.01）. Conclusion Cnidii Fructus hypnotic active components can increase the expression of Cry1, Per1, and Per2 gene by reducing the hypnosis of hippocampal Clock and Bmal1 expression. The increased expression of the inhibitory neurotransmitter GABA and the decreased expression of excitatory neurotransmitter Glu can inhibit the occurrence and development of insomnia, which can regulate the sleep-wake cycle for clinical treatment of insomnia.